Protocols

Histology

Embryos (S2-S8) were fixed for 4 hours at room temperature in 4% formaldehyde in 1x PBS, followed by 3 x 10 minute washes in 1x PBS and gradual dehydration in 30%, 50%, 70%, 80%, 95% and 100% ethanol. The samples were soaked for 30 minutes in 5% glycerol diluted in 100% ethanol, cleared in xylene for 10 minutes, then soaked in two changes of Clear-rite 3 (Richard-Allan Scientific) for a total of 25 minutes. Paraffin infiltration proceeded with 2 x 45 minute incubations, and embedded embryos underwent serial sectioning (5 µm thickness). Paraffin was removed prior to staining by heating slides at 60˚C for 20 minutes, then performing 3 x 2 minute washes in xylene, 3 x 1 minute washes in 100% ethanol, 3 x 1 minute washes in 80% ethanol before rinsing in tap water. Hematoxylin and eosin staining was performed using the ST Infinity H&E Staining System (Leica Biosystems) in a Leica Autostainer. Slides were incubated for 30 seconds in Hemalast, for 2 minutes in hematoxylin, and were rinsed for 2 minutes in tap water. Next, slides were incubated for 45 seconds in differentiator and 1 minute in bluing agent, with each step followed by a 1 minute tap water rinse, and a 1 minute incubation in 80% ethanol. Slides were stained with eosin for 30 seconds, dehydrated 3 x 1 minute in 100% ethanol and cleared in 3 x 1 minute incubations in xylene.

S2-S7 embryos were not bleached. S8 embryos and C4 adults were bleached in formamide bleaching solution for 30 minutes to 1 hour under bright light.

Sexually mature adult worms (≥ 1 cm in length) were treated with 10% N-acetyl cysteine in 1x PBS for 10 minutes, followed by fixation in 4% formaldehyde in PBSTx (0.5%) for 90 minutes. Worms were rinsed in PBSTx (0.5%) for 10 minutes, and then incubated in 10% SDS in 1x PBS for 10 minutes. Reduction time was increased to 20 minutes. Worms were bleached for 2 hours in formamide bleaching solution under bright light.

Microscopy

A Leica M205 FA stereomicroscope was used to capture images of live animals and colorimetric WISH samples. A Leica DM600B upright microscope was used to capture images of histological sections. A Zeiss LSM-510-VIS confocal and a customized light sheet microscope were used to capture Z-stacks for fluorescent WISH samples.

Fixed, stained Smed embryos were mounted in 1% low melt agarose in 1x PBS along with fluorescent conjugated beads required for image registration and reconstruction (FluoSpheres® Polystyrene Microspheres, 1.0 µm, red fluorescent (580/605), Invitrogen/Molecular Probes, F13083; FluoSpheres® Carboxylate Modified Microspheres, 0.1 µm, yellow-green fluorescent (505/515), Invitrogen/Molecular Probes, F8803). 1 µM fluorescent bead stock solutions were diluted 1:10,000 - 1: 360,000, depending on the size of the embryo and the magnification of the detection objective used. Samples were placed in an imaging chamber within a Single Plane Illumination Microscopy (SPIM) system described in (Nakajima et al., 2013). S3-S5 embryos were imaged using either a 10x Plan Apochromat or a 5x Plan NeoFluar objective. Z-stacks were taken every 45˚ around the surface of the samples using a rotating stage, producing 8 stacks of images per embryo. Multiview data sets were reconstructed using Fiji SPIM plugins for data registration and fusion (Preibisch et al., 2010). Completely reconstructed data sets were viewed in the Imaris software package, where they were cropped and masked to remove beads. Cell positions and the embryonic pharynx were marked manually using the 3D Spot Finder function, and the three-dimensional coordinates for marked cells were exported into excel for analysis of cell positions. Colocalization was determined manually on S3-S4 whole embryos, or on crop3D sections (100 µm X 200 µm X 100 µm) of S5 embryos.